Emilio Gil-Martín

Expression and enzyme activity of α(1,6)fucosyltransferase in human colorectal cancer

Changes in enzyme activity and the expression levels of α(1,6)fucosyltransferase [α(1,6)FT] have been reported in certain types of malignant transformations. To develop a better understanding of the role of α(1,6)FT in human colorectal carcinoma (CRC), we analysed the enzyme activity in healthy and tumour tissues. α(1,6)FT activity was considerably higher in tumour tissue than in healthy tissue and was related to gender, lymph node metastasis, type of growth and tumour stage. We also observed a significant increase in the α(1,6)FT expression in tumour tissues as compared to healthy and transitional tissues, inflammatory lesions and adenomas. The immunohistochemical expression in tumour tissues was correlated with the degree of infiltration through the intestinal wall. Finally, a statistical correlation was found between enzyme activity and expression obtained by Western blot in colorectal tumours when compared in the same patient. All these findings demonstrate an alteration of α(1,6)FT activity and expression in CRC.

Correlation Analysis between Tumorous Associated Antigen Sialyl-Tn Expression and ST6GalNAc I Activity in Human Colon Adenocarcinoma

Objectives: Sialyl-Tn (sTn) is a mucin carbohydrate-associated antigen that is strongly expressed in a large number of colorectal carcinomas. In this study, we combined immunohistochemical and enzymatic techniques in order to find the correlation between sTn tissue expression and the sialyltransferase activity (ST6GalNAc I) responsible for its synthesis in colorectal cancer (CRC) patients. Methods: We compared sTn expression in healthy (n = 46), tumorous (n = 60) and transitional tissue (n = 46) from CRC patients, and correlated sTn altered expression with clinicopathologic variables of the patient. Furthermore, we determined ST6GalNAc I tissue activity employing asialo-ovine submaxillary mucin (asialo-OSM) as glycoprotein acceptor (n = 27). Results: The rates of sTn positive expression obtained for healthy, tumorous and transitional tissues were 15, 67 and 63%, respectively. These rates led to statistically significant differences between healthy and tumorous or transitional tissue (p = 0.001); sTn expression was related to the first stages of the tumor invasion in transitional tissue. As regards ST6GalNAc I activity, we found an enhancement in transitional tissue. Statistical correlation analysis did not reveal association between sTn expression and ST6GalNAc I activity. Conclusions: Our findings indicated that sTn antigen tissue expression and ST6GalNAc I activity levels were not correlated in CRC, in spite of the overexpression of the antigen in tumorous and transitional tissue.

Ischemic brain injury: New insights on the protective role of melatonin

Stroke represents one of the most common causes of brains vulnerability for many millions of people worldwide. The plethora of physiopathological events associated with brain ischemia are regulate through multiple signaling pathways leading to the activation of oxidative stress process, Ca2+ dyshomeostasis, mitochondrial dysfunction, proinflammatory mediators, excitotoxicity and/or programmed neuronal cell death. Understanding this cascade of molecular events is mandatory in order to develop new therapeutic strategies for stroke. In this review article, we have highlighted the pleiotropic effects of melatonin to counteract the multiple processes of the ischemic cascade. Additionally, experimental evidence supports its actions to ameliorate ischemic long-term behavioural and neuronal deficits, preserving the functional integrity of the blood-brain barrier, inducing neurogenesis and cell proliferation through receptor-dependent mechanism, as well as improving synaptic transmission. Consequently, the synthesis of melatonin derivatives designed as new multitarget-directed products has focused a great interest in this area. This latter has been reinforced by the low cost of melatonin and its reduced toxicity. Furthermore, its spectrum of usages seems to be wide and with the potential for improving human health. Nevertheless, the molecular and cellular mechanisms underlying melatonin´s actions need to be further exploration and accordingly, new clinical studies should be conducted in human patients with ischemic brain pathologies.

α(1,6)Fucosyltransferase expression is an independent prognostic factor for disease-free survival in colorectal carcinoma ☆

We previously reported that α(1,6)fucosyltransferase (Enzyme class 2.4.1.68) activity and expression are increased in colorectal cancer, suggesting a role for this enzyme in tumor development and progression. However, the possible impact of α(1,6)fucosyltransferase activity or expression on clinical outcomes in colorectal cancer patients has never been studied. Thus, the present study was conducted to determine the value of α(1,6)fucosyltransferase as a prognostic factor for colorectal cancer. α(1,6)Fucosyltransferase expression was analyzed using immunohistochemistry in 141 colorectal tumors, and α(1,6)fucosyltransferase activity was determined in 39 tumors. A complete standardized follow-up of patients was documented until the end of the observation period of 5 years or patient death. Univariate analysis demonstrated the absence of a correlation between enzyme activity and disease evolution. However, in patients with moderate or strong α(1,6)fucosyltransferase expression, a significant decrease in the overall (P = .04) and disease-free (P = .03) survival rates was observed. In addition, when local and distant disease recurrence were considered separately, enzyme expression was found to correlate with local tumor recurrences (P = .01). Furthermore, multivariate analysis showed that α(1,6)fucosyltransferase expression has independent value for predicting tumor recurrences and, specifically, local recurrences. These findings suggest that α(1,6)fucosyltransferase expression may be a good indicator of poor prognosis in colorectal cancer and, therefore, a helpful tool to choose the most effective treatment.

Effect of Human Colorectal Carcinogenesis on the Neural Cell Adhesion Molecule Expression and Polysialylation

Objective: Although downregulation of neural cell adhesion molecule (NCAM) has been correlated with poor prognosis in colorectal cancer (CRC), it is also possible that colon cancer spreading comes from reducing tumor cell adhesion through NCAM polysialylation, as occurs in lung carcinoma or Wilms’ tumor. Methods: To prove this hypothesis, we have performed a prospective study on tumor and control specimens from 39 CRC patients, which were immunostained for NCAM and PSA (polysialic acid) expression. Results: Tumor versus control expression of NCAM and PSA epitopes in tissue specimens, as well as correlation between tumor expression and clinicopathological features, were statistically analyzed. Results showed a low constitutive expression of NCAM and PSA (PSA-NCAM) in control tissue, which reached a statistically significant increase in the tumor tissue. Likewise, the presence and number of lymph node metastases at surgery were correlated with NCAM expression and PSA/NCAM coexpression. Conclusions: These data highlight the importance of taking into account PSA-associated epitopes when dealing with NCAM cell expression studies in tumor development and progression. The analysis of PSA and NCAM expression in CRC suggests a new way, other than downregulation of NCAM, in order to escape contact inhibition and promote cell tumor spreading in colorectal cancer.

Synthesis and expression of CDw75 antigen in human colorectal cancer

BackgroundIncreased ST6Gal I activity has been associated with the α(2,6)sialylation enhancement of membrane glycoconjugates observed in metastatic colorectal carcinomas (CRC). Siaα(2,6)Galβ(1,4)GlcNAc sequence, known as CDw75, is a sialylated carbohydrate determinant generated by the ST6Gal I. This epitope has been reported to be associated with the progression of gastric and colorectal tumours, hence there are only a few conclusive studies to date.MethodsBy radioisotopic techniques we evaluated the ST6Gal I activity in healthy, transitional and tumour tissues from 43 patients with CRC. By immunohistochemistry we assessed the CDw75 expression in 25 colorectal adenomas, 43 tumours, 13 transitional and 28 healthy tissues of CRC patients.ResultsST6Gal I activity was likewise found to be statistically higher in tumour tissue respect to healthy tissue from CRC patients. CDw75 expression was positive in 20% of colorectal adenomas. Furthermore, 70% of tumour specimens and 8.3% of transitional specimens were positive for CDw75 expression, whereas none of the healthy ones showed the presence of the epitope.ConclusionThe major contribution of this study is the inclusion of data from transitional tissue and the analysis of CDw75 antigen expression in CRC and in colorectal adenomas, little known so far. ST6Gal I activity and CDw75 antigen expression were increased in CRC. Although their comparison did not reach the statistical significance, a great extent of patients showed both, an enhanced tumour ST6Gal I activity and an increased CDw75 expression in the tumour tissue. So, these two variables may play a role in malignant transformation. The expression of CDw75 in colorectal adenomas suggests that this antigen may be a tumour marker in CRC.

Elevation of ST6Gal I Activity in Malignant and Transitional Tissue in Human Colorectal Cancer

Objectives: The aim of the present study was to investigate the activity of CMP-NeuAc:Galβ(1,4)GlcNAc sialyltransferase (ST6Gal I) in colorectal cancer (CRC). Methods: ST6Gal I activity was determined in healthy, transitional and tumor tissues from the same patient using asialotransferrin and N-acetyllactosamine as acceptors. Results: ST6Gal I activities with asialotransferrin (n = 85) and N-acetyllactosamine (n = 40) as acceptors were statistically significantly enhanced in CRC tissue compared with healthy mucosa from the same patient (p = 0.001). Using transitional tissue (n = 27), enhancement versus healthy tissue was observed (p < 0.05). A positive correlation was found between ST6Gal I activity with N-acetyllactosamine and asialotransferrin in healthy (n = 32), tumorous (n = 32) and transitional tissue (n = 27), supporting the fact that the same enzyme was detected using both acceptors. Furthermore, we studied the relationship between some patients’ clinicopathological features and ST6Gal I activity. Although the differences were not statistically significant, the levels of ST6Gal I activity in tumorous and transitional tissues varied with the histological grade of the tumor; however, we failed to find a correlation with the AJCC tumor classification. Conclusions: This work reports enhanced ST6Gal I activity in tumor and transitional tissues from CRC patients. However, our overall results suggest that ST6Gal I activity is not indicative of the patient’s outcome.

Identification of α(1,6)fucosylated proteins differentially expressed in human colorectal cancer

SummaryBackgroundA universal hallmark of cancer cells is the change in their glycosylation phenotype. One of the most frequent alterations in the normal glycosylation pattern observed during carcinogenesis is the enhancement of α(1,6)linked fucose residues of glycoproteins, due to the up-regulation of the α(1,6)fucosyltransferase activity. Our previous results demonstrated the specific alteration of this enzyme activity and expression in colorectal cancer, suggesting its implication in tumour development and progression.MethodsIn the current work we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in colorectal cancer. This strategy allowed the identification of a group of α(1,6)fucosylated proteins candidates to be involved in CRC malignancy.ResultsThe majority of the identified proteins take part in cell signaling and interaction processes as well as in modulation of the immunological response. Likewise, we confirmed the increased expression of GRP94 in colorectal cancer tissue and the significant down-regulation of the IgGFcBP expression in tumour cells.ConclusionAll these results validate the importance of core-fucosylated proteins profile analysis to understand the mechanisms which promote cancer onset and progression and to discover new tumour markers or therapeutic targets.

N-Acetyl-β-Hexosaminidase Activity and Isoenzymes in Human Gastric Adenocarcinoma

The enhancement of lysosomal β-hexosaminidase degradative activity in different human cancer tissues is fairly well documented. Gastric tumors have attracted considerable attention on the basis of their social incidence and clinical recurrence. Here we report a comparative study of β-hexosaminidase activity and of its isoenzymes β-hexosaminidase A (HA) and β-hexosaminidase B (HB) from gastric adenocarcinoma and normal mucosa. Tumor β-hexosaminidase activity from crude extracts and chromatographically resolved HA and HB forms were analyzed as regards their physicochemical and enzymatic properties and were compared to similar samples obtained from control tissue. The existence of one active site in the β-hexosaminidase enzyme responsible for both N-acetyl-β-D-glucosaminidase and N-acetyl-β-D- galactosaminidase activities was determined. Apart from their relative contributions to β-hexosaminidase activities, two major differences appeared in tumor HA and HB forms with respect to the corresponding controls: (1) the presence of an atypical heat-stable HB isoenzyme in gastric adenocarcinoma, and (2) a significantly increased Vmax of the HA form acting on both p-nitrophenyl-N-acetyl-β-D-glucosaminide and p-nitrophenyl-N-acetyl-β-D-galactosaminide substrates. The results show that the β-hexosaminidase HA and HB isoenzymes from gastric adenocarcinoma display different patterns of response from the same forms from other human tumors.

Alterations of CMP-NeuAc:asialofetuin sialyltransferase activities in human colorectal adenocarcinoma.

It has been reported that surface glycoconjugates in tumour cells have more complex and more heavily sialylated sugar chains in glycoproteins. Here, we analysed CMP-NeuAc:asialofetuin sialyltransferase activities in total cell membranes from colorectal cancer tissue with respect to normal adjacent tissue, finding enhanced sialyltransferase activities. Determination of the kinetic parameters for the donor substrate, CMP-NeuAc, revealed that the apparent Km in tumour tissue was decreased as regards to the normal value. Furthermore, we failed to find any correlation between this activity and the characteristics of the samples such as the age or sex of the patient, or Dukes’ stage of the tumour. In order to know which sialyltransferase activity was altered, we studied the incorporation of NeuAc into N- and O-linked chains from asialofetuin. The results allow us to conclude that it is on N-linked chains where the activity is enhanced. With respect to α(2,3)- and α(2,6)-NeuAc linkages, we observed that enhanced activity in tumour tissues affects both linkages equally.